Multiwell microelectrode array with optical stimulation

ABSTRACT

An electro-optical stimulation and recording system is disclosed, including a substrate and a plurality of wells coupled to the substrate. The system also includes at least one electrode set disposed proximate a respective one of the plurality of wells, wherein the electrode set comprises at least one electrode configured to collect an electric signal associated with at least a portion of the respective well. The system also includes a light-emitting element set corresponding to a respective one of the wells and configured to deliver optical stimulation to at least a portion of the respective well.

TECHNICAL FIELD

The present disclosure relates generally to tools for facilitatingoptogenetic stimulation of cells and tissue and, more particularly, tomultiwell microelectrode arrays with optical stimulation capabilitiesand associated methods for using the same.

BACKGROUND

Microelectrode arrays (MEAs) are an invaluable tool for scientificdiscovery and medical research. Because they can actively monitor andmanipulate cellular activity (at both the single-cell and tissue levels)using electrical stimulation/recording, MEAs provide extraordinaryinsight into cell network interactions. Many conventional MEAs are ofthe single-well variety, meaning that only a single cell or tissueculture may be tested/analyzed at a time. Consequently, testing ofmultiple cell or tissue samples using conventional single-wellmicroplates typically requires a significant monetary investment inmultiple single-well measurement test beds, a significant allocation oftime to sequentially test each cell or tissue sample, or somecombination of the two.

To provide a more cost- and time-efficient platform for simultaneouslytesting multiple cell or tissue cultures, multiwell MEAs were developed.Unlike their single-well counterparts, multiwell MEAs provide an arrayof culture wells, each of which has a corresponding array of electrodesfor recording electrical activity from (and/or delivering electricalstimulation to) the contents of the well. Current multiwell MEAs come ina variety of sizes, including, 4-, 12-, 24-, 48-, 72-, 96-, and 384-wellconfigurations, providing a significant number of options for scaling invitro testing to meet the needs of most any experimental setting.Although multiwell MEAs have certainly alleviated the scalabilityproblems associated with single-well MEAs, they are generally limited intheir ability to deliver different modes of stimulation (e.g.,electrical, optical, thermal, etc.)

More specifically, despite the relative success of multiwell MEAsystems, the technology's impact may be limited by the inherentlimitations of electrical stimulation. Electrical stimulation pulsesfrom MEA microelectrodes are limited to the locations of the electrodesand excite all nearby electroactive cells, regardless of cell sub-type.Electrically mediated inhibition of cell activity requires complexstimulation paradigms that are impractical and unreliable. Additionally,the amount of charge injection required for extracellular stimulationcan saturate sensitive electronics and leave residual charge on theelectrodes. In turn, this charge creates blind spots in electricalrecordings that obscure critical activity around the time ofstimulation. Therefore, there is a need for new stimulation solutionsthat can more selectively control cell networks without creatingdistortions or artifacts in the electrophysiological recordings.

Optogenetic stimulation techniques provide a more selective mechanismfor manipulating cell cultures. In optogenetics methodologies, selectedcells are genetically manipulated to express light sensitive membraneproteins called opsins. Specific cell types within heterogeneouscultures can then be genetically targeted for activation or inhibitionwith light of specific wavelengths. This light can be precisely pulsedand more evenly delivered across cultures, stimulating (or inhibiting)only the targeted cell types, while creating minimal stimulationartifact. Using different methodologies, optogenetic stimulation canalternatively provide the capability to influence intracellularsignaling.

In order to provide a multiwell MEA solution with enhanced capabilityfor selectively targeting different types of cells within a culture ortissue sample, a multiwell MEA system with integrated, independentlycontrollable optical stimulation capabilities would be advantageous. Thepresently disclosed multiwell microelectrode arrays with integratedoptical stimulation capabilities and associated methods for using thesame are directed to overcoming one or more of the problems set forthabove and/or other problems in the art.

SUMMARY

According to one aspect, the present disclosure is directed to anelectro-optical stimulation and recording system, comprising a substrateand a plurality of wells coupled to the substrate. The multiwell platemay also include one or more electrode sets, each electrode set disposedproximate a respective one of the plurality of wells. Each electrode setcomprises at least one electrode configured to collect an electricsignal associated with at least a portion of the respective well. Themultiwell plate may also include at least one light-emitting element setcorresponding to a respective one of the wells and configured to deliveroptical stimulation to at least a portion of the respective well.

In accordance with another aspect, the present disclosure is directed toa method for large-scale in vitro testing or manipulation of cellcultures. The method may comprise providing a control signal for causinga light-emitting element to emit light in an illumination pattern, thelight-emitting element disposed proximate a well of a multiwell plate.The method may also comprise detecting, via an electrode disposedproximate the well, a signal associated with at least a portion of thewell. The method may also comprise analyzing the detected signal andoutputting information indicative of the analysis. The method may alsocomprise comparing the detected signal with benchmark data. The methodmay also comprise determining, based on the comparison, that adjustmentof the illumination pattern is required. The method may also comprisemodifying one the illumination pattern based on the determination. Thedetected signals may be analyzed, and information indicative of theanalysis may be output, via a user interface element on a display.Optical stimulation parameters may be automatically adjusted based ondetected signals, on a per well basis. Such automatic, algorithmicadjustment may be useful, for example, to optimally adjust lightpatterns on a per well basis. Software tools may allow light patterns tobe generated and directed to selected wells and provide visualization ofdelivered light alongside visualizations of detected electricalactivity.

In accordance with another aspect, the present disclosure is directed toa an electro-optical stimulation and recording system, comprising anelectrode set disposed proximate a first well and comprising a firstelectrode configured to collect an electric signal associated with atleast a portion of the first well. The system may also comprise a firstlight-emitting element set configured to deliver optical stimulation toat least a portion of the first well; and a processor. The processor maybe configured to provide a first control signal for causing the firstlight-emitting element to emit light at a first illumination pattern;and detect, via the first electrode, a first signal associated with atleast a portion of the respective well.

In another example embodiment, the present disclosure is directed to anoptical stimulation system comprising a microplate having a plurality ofwell. The system may also comprise at least one light-emitting elementset corresponding to at least one of the plurality of wells andconfigured to deliver optical stimulation to at the at least one well.The system may also comprise a lid configured to couple to themicroplate, wherein the lid enhances delivery of light through the lidvia at least one of recesses, lenses, and reflective surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example system having a multiwell microelectrodearray with optical stimulation delivery capability for controllablestimulation of culture wells, in accordance with certain disclosedembodiments.

FIG. 2 provides a detail view of an optical stimulation control anddelivery portion of the system illustrated in FIG. 1, consistent withcertain disclosed embodiments.

FIG. 3A provides a diagrammatic perspective view of an example 48-wellmicroelectrode array (MEA), consistent with certain disclosedembodiments.

FIG. 3B provides a zoomed diagrammatic perspective view of a selectionof wells of a 48-well microelectrode array (MEA), in accordance withcertain disclosed embodiments.

FIG. 4 provides a diagrammatic perspective view of a multiwellmicroelectrode array (MEA) with a detachable optical stimulation modulecoupled to the MEA, consistent with certain disclosed embodiments.

FIG. 5 provides a schematic block diagram of a cross-section of amultiwell microelectrode array (MEA) with a detachable opticalstimulation module coupled to the MEA, in accordance with certaindisclosed embodiments.

FIG. 6 provides a schematic block diagram of a cross-section of anotherembodiment of multiwell microelectrode array (MEA) with a detachableoptical stimulation module coupled to the MEA, consistent with certaindisclosed embodiments.

FIG. 7A provides an overhead (top) view of an exemplary single well(i.e., corresponding to a single culture well of a multiwell MEA) 8×8grid of electrodes used in the presently disclosed multiwellmicroelectrode array (MEA) with optical stimulation capabilities, inaccordance with certain disclosed embodiments.

FIGS. 7B and 7C provide detail overhead (top) zoom views of an exemplarysingle (i.e., corresponding to a single culture well of the multiwellMEA) 8-microelectrode array used in the presently disclosed multiwellmicroelectrode array (MEA) with optical stimulation capabilities,consistent with certain disclosed embodiments.

FIG. 8 provides a block diagram of a processor-based computing systemhaving an integrated data analysis and control suite that is used tocontrol and conduct various experiments using the presently disclosedmultiwell microelectrode array (MEA) with optical stimulationcapabilities, in accordance with certain disclosed embodiments.

FIGS. 9A, 9B, 9C, 9D, and 9E provide screen shots associated with anexample user interface associated with the integrated data analysis andcontrol suite that is used to conduct and visualize various experimentsthat may be performed using the presently disclosed multiwellmicroelectrode array (MEA) with optical stimulation capabilities,consistent with certain disclosed embodiments.

FIG. 10 illustrates an exemplary multiwell microelectrode array systemcontaining a 48-well multiwell microelectrode array plate, and adetachable optical stimulation device for optically stimulatingindividual culture wells, in accordance with certain disclosedembodiments.

FIG. 11 provides a schematic block diagram of certain componentsassociated with the exemplary interface system illustrated in FIG. 10and its corresponding interactions between the multiwell MEA plate 300and control and monitoring system 800.

FIG. 12 provides a functional block diagram associated with an exemplaryprocess for use and control of the presently disclosed multiwellmicroelectrode array (MEA) with optical stimulation capabilities,consistent with certain disclosed embodiments.

FIG. 13 provides a diagrammatic perspective view of an individualculture well within an exemplary multiwell microelectrode array (MEA)plate, along with an overlying detachable optical stimulation module,consistent with certain disclosed embodiments.

FIGS. 14A, 14B, and 14C provide diagrammatic cross-sections ofindividual culture wells in different example multiwell microelectrodearray (MEA) plates, along with overlying detachable optical stimulationmodules, shown coupled to the MEA plates. The multiwell MEA plate lidsand LED reflectors in FIGS. 14A, 14B, and 14C are specialized to enhanceoptical delivery and/or prevent the passage of light between adjacentwells, consistent with certain disclosed embodiments.

DETAILED DESCRIPTION

FIG. 1 illustrates an exemplary electro-optical stimulation deliverysystem 100 having a multiwell microelectrode array and opticalstimulation delivery capability in accordance with certain disclosedembodiments. The electro-optical stimulation delivery system 100illustrated in FIG. 1 may provide a platform for controlling delivery ofoptical (and, in some cases, electrical) stimulation to a subset ofculture wells and actively monitoring the electrical activity associatedwith the cells contained in the well. The electro-optical stimulationdelivery system 100 may include, among other things, an opticalstimulation module 210, a multiwell microelectrode array 300, and an MEAplate interface 1000.

Optical stimulation module 210 is also referred to herein as “opticalstimulation device.” In some embodiments, an optical stimulation modulemay be integrated with the MEA plate. In other example embodiments, anoptical stimulation module may be a standalone device that detachablycouples to an MEA plate. For example, the optical stimulation module maycouple to the top of the MEA plate or may be coupled to the bottom ofthe MEA plate. Other configurations are possible as well and thelanguage herein is not intended to limit the possible implementations ofthe optical stimulation module.

Those skilled in the art will recognize that the components describedabove with respect to electro-optical stimulation delivery system 100are exemplary only and not intended to be limiting. Consequently,electro-optical stimulation delivery system 100 may include additional,and/or different components than those shown in FIG. 1 without departingfrom the scope of the present disclosure. Each of the individualcomponents of electro-optical stimulation delivery system 100 shown inthe embodiment illustrated in FIG. 1 will be described in greater detailbelow.

Although FIG. 1 illustrates optical stimulation module 210 and multiwellMEA plate 300 as separate elements, it is contemplated that one or moreof the features of optical stimulation module 210 may be included aspart of multiwell MEA plate 300 (or vice versa). Indeed, certainembodiments contemplate a multiwell microelectrode array with integratedoptical stimulation capabilities, regardless of whether the opticalstimulation capabilities are provided by a separate, standalone system(as shown in FIG. 1), or by an integrated solution as part of amultiwell MEA plate 300.

FIG. 2 illustrates a schematic block diagram of an optical stimulationdelivery system 200. Optical stimulation delivery system 200 includes aplurality of components that control the delivery of optical stimulationto MEA plate 300. As illustrated in FIG. 2, and in accordance with oneembodiment, optical stimulation delivery system 200 may include anoptical stimulation module 210 and a control and monitoring system 800that is programmed to control optical stimulation module 210.

Optical stimulation module 210 may include one or more light-emittingelement sets 230 that may be optimized/customized to meet most any powerand thermal criteria that may be required. Optical stimulation module210 may also include control circuitry 220 for independently controllingthe light-emitting elements with light patterns of high temporalresolution, as well as finely graded control over output intensity. Thesystem may also include interactive software that will enable generationof customizable stimulation waveforms, and visualization of thesedelivered stimulation waveforms. As such, the presently disclosed systemmay be configured to manage cell-targeted activation or inhibition formultiple cultures in a multiwell MEA. The term “light-emitting elementset” is not intended to limit the number of light-emitting elementswithin a given “set.” For example, a light-emitting element set maycomprise one, or multiple, light-emitting elements.

As shown in FIG. 2, each of light-emitting element sets 230 may includeor embody any suitable component for delivering optical energy to arespective culture well with which the light-emitting element isassociated. According to one embodiment, (and as illustrated in FIG. 4),light emitting diodes (LEDs) may be provided in an overlying orlid-based array (at least one LED per well). This configuration isparticularly advantageous in situations where a compact, top-side formfactor may be required. Importantly, however, although certainembodiments call for a single LED per well, it is certainly contemplatedthat light-emitting element sets may include multiple light-emittingelements per well (as illustrated, for example, in FIG. 6). It iscontemplated that, although LEDs are described in certain exemplaryembodiments as the light-emitting elements, any of a number oflight-emitting sources may be adapted for implementation. For example,light-emitting elements may include or embody any (or a combination) oflasers, organic light-emitting diodes (OLEDs), 2D pixel displays,digital light projection (DLP) technology, a waveguide element, alight-emitting carbon nanotube, or any other element suitable forproviding a controllable light source for delivering optical energy tothe culture well.

As illustrated in FIG. 2, each light-emitting element 230 may be coupledto control and monitoring system 800 via control circuitry 220.Light-emitting elements 230 may be controlled by any of a number ofcommercially available control modules. In embodiments that implementLEDs as the light-emitting elements, current-control modules (e.g.,RCD24-1.0, RECOM) may be used. Such circuits may be used to control highintensity LEDs (e.g., Oslon SSL, Osram Opto Semiconductors) of one ormore wavelengths. Light-emitting elements 230 may be configured todeliver one or more of infrared light energy, ultraviolet light energy,or visible light energy, depending upon the type of experiment beingperformed (and/or the type of light-reactive opsin being targeted, inthe case of optogenetics applications).

Control circuitry 220 for the optical stimulation device may beconfigured to independently control individual light-emitting elementsor may control sets of multiple light-emitting elements as a group.System control may be handled by a 32-bit microcontroller (e.g., MCF5213; Freescale). Each LED in the array might be driven by an RCD24-1.0LED Driver. This driver may control LED intensity with an analog inputsignal, which may be set by the microcontroller through a high channelcount digital to analog converter (AD5391, Analog Devices). The drivermay also take a digital on/off signal for pulsed or triggered waveforms.The microcontroller may be programmed via a high speed USB-to-UART/FIFO(FT2232H, FTDI) chip.

According to an exemplary embodiment, light-emitting elements 230 may bepositioned above individual wells on an n-well MEA plate and drivencontinuously or modulated at a fixed or variable current duringelectrical recordings. The presently disclosed multiwell microelectrodearray with coupled optical stimulation module may be configured toreduce both steady-state and transient noise caused by thelight-emitting elements. Steady-state electrical noise may be eliminatedwith proper grounding of the LED driver circuitry. Electrically-inducedtransient noise may be reduced/eliminated by placing a transparent,electrically conductive, grounded layer (e.g., indium tin oxide (ITO))between the LED and MEA well. When present, any photoelectric artifactmay be reduced with, for example, typical bandpass filtering used forrecordings of electroactive cells (e.g. 200-3000 Hz bandpass filter forspike signals (e.g., neural or cardiac)).

As explained, electro-optical stimulation delivery system 100 comprisesone or more components that cooperate to deliver optical (and, in someembodiments, electrical) stimulation to one or more of a plurality ofculture wells in a multiwell microelectrode array device 300 andsimultaneously detect electrical signals from the culture wells,including those signals that are indicative of the cellular response tothe stimulation FIGS. 3A and 3B each provide a diagrammatic perspectiveview of the structural components of a multiwell culture array 300. Asillustrated in FIGS. 3A and 3B, a multiwell culture device (regardlessof whether it is configured as a multiwell MEA or multiwell MEA withoptical stimulation capabilities) typically includes a plurality ofculture wells 310 disposed upon substrate 312. FIG. 3A illustratesmultiwell array 300 as a 48-well device. It is contemplated, however,that any number of wells may be provided without departing from thescope of the present disclosure. Furthermore, the dimensions shown inFIG. 3A are exemplary only, and not intended to be limiting. Accordingto an exemplary embodiment, the presently disclosed multiwell arrays maybe sized and configured in an ANSI-SLAS compliant format, compatiblewith traditional plate readers and automated instrumentation.

Multiwell MEA 300 includes a matching lid designed for maintainingsterility and reducing media evaporation. Each culture well comprises aninternal volume for receiving cell culture material at an opening in thetop of the well. In one embodiment, cells can be added within a smalldrop to the center of the well (where the MEA electrodes reside). Afterthe cells have an opportunity to attach to the substrate, then the wellcan be flooded with more cell culture medium. Electrical signalsresulting from cellular reactions can be monitored by electrodespositioned in the bottom of culture well. Electrical connections forconnecting the electrodes to MEA plate interface 1000 are located on thebottom-side of the device. It is contemplated that the multiwell MEAmight be used along with other biological, environmental, or chemicalsamples aside from cell cultures.

As shown in FIG. 3B, each culture well 310 may embody a receptacle forreceiving cell culture and/or tissue material through an opening at thetop of the well. According to one embodiment, the walls 311 and/orsubstrate and/or lid of culture well 310 may be comprised of or coatedwith reflective material to maximize the light delivery to the base ofthe well and to prevent light absorption or loss through the wall of thewell. According to one embodiment, reflective white-walled multiwellplates may be used to increase light delivery by reflecting andconcentrating light within the wells, while blocking passage of light toadjacent wells. Additionally, light delivery may be increased throughthe use of commercially available clear media, as culture media dyedwith Phenol Red was found to significantly absorb blue and green light.Electrodes (not shown) positioned at the base 312 of the culture well310 can be configured to provide electrical stimulation and monitor anyelectrical cellular, biological, or chemical activity or cellular,biological, or chemical sample properties within a given well.

FIGS. 4, 5, and 6 illustrate an exemplary configuration of the multiwellmicroelectrode array 300 with coupled optical stimulation module 210consistent with the disclosed embodiments. Typical mid- to high-powerLEDs can draw high current loads and dissipate significant heat. Forexample, a high power 465 nm blue LED might consume 1 W power anddissipate 0.5 W of heat when run at a moderate supply current of 350 mA.These types of power and thermal costs scale with LED count, requiredlight intensity, and stimulation duty cycle. Consequently, the presentlydisclosed system includes robust power supply circuitry 410 withdedicated control of the current delivered to each LED (230A, 230B,230N). As shown in FIGS. 4 and 5, multiwell MEA 300 may include one ormore components for managing heat dissipation to ensure efficient LEDoperation, prevent LED device damage, and reduce the amount of heatdelivered to cell cultures. The system may utilize metal core printedcircuit boards and, in some embodiments, a custom aluminum or copperheat sink 420 with an optional active cooling system. Although FIGS. 4and 5 illustrate the optical stimulation module 210 having a single LEDper set, it is contemplated that each set may include a plurality ofindependently controllable light emitting elements 650 configured todeliver multiple wavelengths of light to each well.

FIG. 6 illustrates an exemplary multiwell microelectrode array with acoupled optical stimulation module, where each light-emitting elementset includes a plurality of light-emitting components 650. Eachlight-emitting component may be configured to deliver a differentwavelength and/or intensity of light to provide greater flexibility inthe types of experiments that can be performed. In addition to multiplelight-emitting elements, the multiwell MEA 300 may also include one ormore devices for enhancing the delivery of optical energy to the culturewell. For example, as illustrated in FIG. 6, multiwell MEA 300 mayinclude one or more light-collimating reflectors 640 for directing theoptical energy to the center of the culture well. Alternatively oradditionally, multiwell MEA 300 may also include other components, suchas lenses, filters, gratings, and other components for modifying theoptical properties depending upon the specific needs of the experiment.These features might be incorporated into the lid 610 of the multiwellplate. For example, lenses and recesses can be inexpensively molded intoa low-cost disposable multiwell plate lid.

As also illustrated in FIG. 6, the optical stimulation module 210 may beconfigured to overlay a transparent lid 610 associated with themultiwell MEA 300. This configuration may allow the optical stimulationmodule 210 to be coupled and de-coupled from multiwell MEA 300, withoutpotentially contaminating the cultures during testing. In thisconfiguration, optical stimulation module 210 may be designed toremovably couple to/from lid 610. As an alternative to the opticalstimulation module 210 being removably coupled to a separate lid, it iscontemplated that optical stimulation module 210 may be integrated withlid 610, or constructed to be the lid for the multiwell MEA 300.

Although FIG. 6 illustrates light-emitting elements that are configuredfor positioning at the opening of (i.e., above) multiwell MEA 300, it iscontemplated that the light-emitting elements may be embedded in acircuit board beneath a transparent floor of the respective wells.According to one exemplary embodiment, light emitting elements may beprovided beneath or within substrate 620 upon which the grid ofelectrodes is disposed. In this embodiment, the electrodes may beprinted on a transparent substrate (e.g., Printed Circuit Board (PCB),flex circuit, or transparent biosensor array) through which light canpass. According to other embodiments, light emitting elements may beincluded as part of the same transparent substrates upon whichelectrodes are disposed. Organic Light Emitting Diode (OLED) technologycan be coupled with micro-biosensor fabrication technologies to achievesuch an integrated transparent OLED-MEA substrate.

According to additional and/or different embodiments, the multiwell MEA300 may also include enhancements to facilitate efficientconservation/delivery of light emitted by optical stimulation module210. For example, multiwell MEA 300 may include strategically shaped orcurved walls 660 to ensure more concentrated light delivery toward thecentral portion of the bottom of the well. Alternatively oradditionally, multiwell MEA may include a reflective material 630,disposed beneath each well and configured to reflect light that istransmitted through the bottom of the well back into the well (in thecase of a transparent substrate 620). The MEA lid 610 may alsoincorporate modifications to increase, enhance, shape, or otherwiseinfluence light delivery to the MEA culture. For example, lenses mightbe molded into the lid to collimate or refract light to the center ofthe well. A Fresnel lens design might accomplish this while extendingminimally into the well. The light might have recesses to allow lightsources such as LEDs or optical fibers to extend some depth into thewell, increasing light delivery, or directing light to one or moreparticular regions of the culture. To minimize condensation that mightcollect on the lid in the course of typical use, possibly interferingwith light delivery, a system of heating the lid might be employed. Inone example, a transparent ITO layer, might be used to heat the lid andminimize condensation. In the case of bottom-side light delivery, thelid might be reflective, to re-direct and concentrate light back intothe culture well.

As explained, multiwell MEA 300 includes one or more electrodes, each ofwhich is configured to measure electrical activity in the surroundingarea. Multiwell MEA 300 includes one or more electrode sets, eachelectrode set comprising one or more (e.g., 8) electrodes, with each setdisposed between the substrate 312 and the base of a respective culturewell that it is configured to monitor. The term “electrode set” is notmeant to limit the number of electrodes in the “set.” For example, anelectrode set may comprise a single electrode or multiple electrodes.

FIG. 7A illustrates a schematic of an exemplary electrode array. Asshown in FIG. 7A, electrode arrays are made up of a grid of tightlyspaced electrodes, and each electrode is capable of simultaneouslymonitoring the activity of individual cells. As illustrated in FIG. 7A,the arrangement of multiple electrodes in a grid extends the recordingrange across a relatively large area, providing concurrent access toboth single cell and tissue- or network-level activity. The control andmonitoring of this cellular activity is made possible by theelectronics, which impart multiple functions to each electrode.

Each electrode of the array facilitates monitoring of single-cell andnetwork-level activity for extended periods of time, with virtually nodestructive interference to the tissue being investigated. In fact, thebroad access to network information, along with the minimally invasivenature of the device, is precisely what makes the MEA an exceptionalsingle-cell and network-level research tool. Each electrode in the highthroughput MEA is ideally suited for investigation of electroactivecells and tissue (e.g., neural, cardiac, muscle, and spinal tissue). Asexplained previously, the MEA-wells are organized in an ANSI-SLAScompliant format, compatible with traditional plate readers andautomated instrumentation. Within each well, a plurality (e.g., between4 and 64 individual embedded microelectrodes (˜30-50 μm diameter;˜200-350 μm center-to-center spacing, in accordance with an exemplaryembodiment) with integrated ground electrodes are capable ofsimultaneously monitoring the activity of individual cells. Thearrangement of these electrodes into a grid extends the recording rangeup to a 1.5×1.5 mm area, providing concurrent access to both single-celland network-level activity.

FIGS. 7B and 7C provide more detailed views (i.e., zoom) views of eachelectrode set associated with an embodiment of the multiwell MEA 300consistent with the present disclosure. According to one embodiment,each electrode is constructed of nano-textured gold on an FR4 epoxyresin with an optional reflective white overlay to reflect light backinto the culture well for increased irradiance of the cell cultures.Nano-texturing of gold is achieved through proprietary processesdesigned to increase the surface area of gold, thereby lowering theelectrode impedance and noise.

Each of the electrodes in every set of one or more electrodes may beconfigured to simultaneously provide stimulation to the culture well andrecord electric signal resulting from stimulation of the cells.Alternatively or additionally, some of the electrodes in the set may beconfigured to provide stimulation only, while other electrodes arededicated to recording cellular activity.

As explained, processes and methods consistent with the disclosedembodiments provide solutions for integrating optical stimulation in amultiwell MEA 300. In addition to the structural and functional aspectof the multiwell MEA 300, the present disclosure is directed toprocesses and methods for using the multiwell MEA 300 to performhigh-throughput, large scale testing, such as testing based onoptogenetics techniques. Consequently, the presently disclosedelectro-optical stimulation delivery system 100 includes a computersystem (or stimulation GUI) that has been customized to control thestimulation of the culture wells and provide an interface forcollecting/analyzing the cellular activity resulting from thestimulation.

FIG. 8 illustrates an exemplary schematic diagram associated with thecontrol and monitoring system 800 that is adapted to interface with amultiwell microelectrode array (MEA) with optical stimulationcapabilities. As explained, control and monitoring system 800 mayinclude processor-based device that includes its own microcontroller,volatile and non-volatile memory, one or more databases, and one or moreinterfaces for communicating data with a user.

According to one embodiment, control and monitoring system 800 mayinclude one or more hardware components including, for example, acentral processing unit (CPU) or microprocessor 811, a random accessmemory (RAM) module 812, a read-only memory (ROM) module 813, a memoryor data storage module 814, a database 815, one or more input/output(I/O) devices 816, and an interface 817. Alternatively and/oradditionally, control and monitoring system 800 may include one or moresoftware media components such as, for example, a computer-readablemedium including computer-executable instructions for performing methodsconsistent with certain disclosed embodiments. It is contemplated thatone or more of the hardware components listed above may be implementedusing software. For example, storage 814 may include a softwarepartition associated with one or more other hardware components ofcontrol and monitoring system 800. Control and monitoring system 800 mayinclude additional, fewer, and/or different components than those listedabove. It is understood that the components listed above are exemplaryonly and not intended to be limiting.

CPU 811 may include one or more processors, each configured to executeinstructions and process data to perform one or more functionsassociated with control and monitoring system 800. As illustrated inFIG. 8, CPU 811 may be communicatively coupled to RAM 812, ROM 813,storage 814, database 815, I/O devices 816, and interface 817. CPU 811may be configured to execute sequences of computer program instructionsto perform various processes, which will be described in detail below.The computer program instructions may be loaded into RAM 812 forexecution by CPU 811.

RAM 812 and ROM 813 may each include one or more devices for storinginformation associated with an operation of control and monitoringsystem 800 and/or CPU 811. For example, ROM 813 may include a memorydevice configured to access and store information associated withcontrol and monitoring system 800, including, for example, stimulationschemes for different types of experiments. RAM 812 may include a memorydevice for storing data associated with one or more operations of CPU811. For example, ROM 303 may load instructions into RAM 302 forexecution by CPU 811.

Storage 814 may include any type of mass storage device configured tostore information that CPU 811 may need to perform processes consistentwith the disclosed embodiments. For example, storage 814 may include oneor more magnetic and/or optical disk devices, such as hard drives,CD-ROMs, DVD-ROMs, or any other type of mass media device. Alternativelyor additionally, storage 814 may include flash memory mass media storageor other semiconductor-based storage medium. Database 815 may includeone or more software and/or hardware components that cooperate to store,organize, sort, filter, and/or arrange data used by control andmonitoring system 800 and/or CPU 811.

I/O devices 816 may include one or more components configured tocommunicate information with a component or user associated with controland monitoring system 800. For example, I/O devices 816 may include aconsole with an integrated keyboard and mouse to allow a user to inputparameters associated with control and monitoring system 800. I/Odevices 816 may also include a display including a graphical userinterface (GUI) for providing a network management console for networkadministrators to configure control and monitoring system 800. I/Odevices 816 may also include peripheral devices such as, for example, aprinter for printing information associated with control and monitoringsystem 800, a user-accessible disk drive (e.g., a USB port, a floppy,CD-ROM, or DVD-ROM drive, etc.) to allow a user to input data stored ona portable media device, a microphone, a speaker system, or any othersuitable type of interface device. I/O devices may be configured tooutput network analysis results and traffic characteristics.

Interface 817 may include one or more components configured to transmitand receive data via a communication network, such as the Internet, alocal area network, a workstation peer-to-peer network, a direct linknetwork, a wireless network, or any other suitable communicationplatform. For example, interface 817 may include one or more modulators,demodulators, multiplexers, demultiplexers, network communicationdevices, wireless devices, antennas, modems, and any other type ofdevice configured to enable data communication via a communicationnetwork. According to one embodiment, interface 817 may be coupled to orinclude wireless communication devices, such as a module or modulesconfigured to transmit information wirelessly using Wi-Fi or Bluetoothwireless protocols.

FIGS. 9A, 9B, 9C, 9D, and 9E provide exemplary user interface screensshots associated with control and monitoring system 800. According toone embodiment, the software may be Axion's Integrated Studio (AxIS)suite of software provided by Axion BioSystems of Atlanta, Ga. Thissoftware may provide an interface that allows users to createsophisticated stimulation experiments via I/O devices 816 and/orinterface 817. This software allows concurrent monitoring of channelrecordings, digital and analog filter adjustments, electrode assignment,and stimulus waveform design, all within the same application in amodular layout.

Software associated with stimulation GUI provides an interface thatallows users to control the stimulation parameters associated with theelectrodes and light-emitting elements. FIG. 9A illustrates an exemplarysoftware interface for independently controlling the stimulationparameters for each culture well. The software interface may also allowthe user to establish and control stimulation parameters 900 associatedwith different groups of target culture wells 910. According to oneembodiment, the user can control the current level and waveform typeassociated with electrical stimulation. Alternatively or additionally,the software interface may allow a user to control the opticalstimulation parameters, such as frequency and amplitude of opticalradiation.

Software associated with stimulation GUI may provide an interface formonitoring real-time experiment data. FIG. 9B illustrates a screen shotof a software recording module that displays a real-time, orpost-experiment, scrolling “raster” plot display of action potentialsdetected by each electrode in a sample MEA well. The timing of eachdetected action potential signal is demarcated by a vertical line 920for each electrode in a sample MEA well. A depiction of deliveredoptical stimulation patterns of one 930 or more wavelengths 940 is shownon the same display. This conceptual example illustrates one way thatdelivered light stimulation might be visualized, and furthermorevisualized in a way that is temporally aligned with electricalrecordings and/or electrical stimulation from the MEA electrodes.

FIG. 9C provides an exemplary, conceptual software visualizationillustrating metrics of detected signals that are further classified andvisualized as light-evoked. In this example, beating from a cardiac cellculture is detected and analyzed, and the timing between consecutivebeats, termed the beat period, is calculated and displayed as real-timebeats 950. If the beats are classified as being evoked by deliveredlight, they are represented differently, as illustrated by beats 960, toallow the experimenter to visually assess the effects of opticalstimulation.

FIG. 9D provides another exemplary conceptual software visualization,whereby an activity metric is calculated and displayed in relation todelivered optical stimulation. In this example, a peristimulus timehistogram of detected action potentials in relation to delivered pulsesof light is generated and continually updated. Indication 980 indicatesthe timing of light delivery and the histogram 970 includes actionpotential counts falling within bins of elapsed time after eachdelivered light pulse.

FIG. 9E provides another exemplary conceptual software visualization,whereby electrical activity 990 detected by each recording electrode isdisplayed in real-time or after an experiment. Characteristics ofdelivered light patterns, including the timing, intensity, or wavelength995, is/are indicated graphically and superimposed on the plots ofdetected activity. The optical stimulation patterns are displayed in amanner that is time synchronized with the visualization of detectedelectrical activity. Such representations allow the experimenter toeasily visually assess the impact of optical stimulation on detectedsignals.

FIG. 10 illustrates an exemplary interface system for communicativelycoupling the presently disclosed multiwell microelectrode array (MEA)with optical stimulation capabilities with a processor-based computingsystem having an integrated data analysis and control suite. An opticalstimulation module 1000 is shown here as a detachable platform that canbe placed onto the multiwell MEA plate 1010 while the plate is docked inthe interface system 1020.

FIG. 11 provides a schematic block diagram of certain componentsassociated with the exemplary interface system illustrated in FIG. 10and its corresponding interactions between the multiwell MEA 300 andcontrol and monitoring system 800. Such an interface system may providefull stimulation and recording access to a plate with 768 electrodes.Three banks of 64-channel ICs interface the electrodes and multiplex theresulting signals by a factor of 8. Additional multiplexing is used tointerface to a bank of ADCs, with FPGAs providing data communication,system coordination, and data processing functions.

FIG. 12 provides a functional block diagram associated with an exemplaryprocess for use and control of the presently disclosed multiwellmicroelectrode array (MEA) with optical stimulation capabilities.Optical patterns 1210 are delivered to one or more MEA wells.Subsequently or simultaneously, electrical signals are detected 1220 byone or more electrodes in one or more MEA wells. These detected signalsare analyzed 1230 and output 1240 by the system. Optionally, theillumination pattern may be adjusted 1250, either by the experimenter orautomatically by the optical stimulation module or processor, based onthe analysis of detected signals 1230. For example, the intensity ofdelivered light pulses might be automatically adjusted on a per-wellbasis to an optimal level 1260, based on detected metrics of neural orcardiac cell culture activity. This feedback-driven tuning of deliveredlight might allow for advanced, algorithmic control over the state ofcell cultures on a per well basis, allowing for advanced control andanalysis of target cells and cell networks.

FIG. 13 provides a conceptual, perspective view of a sectioned MEA wellwithin a multiwell MEA plate 300. In this embodiment, a transparent MEAplate lid 1330 remains on the MEA plate during use. A light deliveryplatform, including an array of LEDs 1310 on an electronics board 1300substrate, and an array of reflectors 1320 or lenses fits onto themultiwell plate. Recesses in the plate lid allow for mechanicalalignment, reduce the escape of light into adjacent wells, and improvethe optics of light delivery. Light passes through a volume of cellculture medium 1350 which might optionally be transparent in color orcolored such that the delivered light is not absorbed by the medium.Lid, reflector, and MEA plate modifications may be incorporated for thepurpose of maximizing efficiency and/or providing uniform light deliveryto the central target cell culture region 1360 above the grid ofmicroelectrodes.

FIG. 14A provides alternate configurations for the lid 1400, reflector1440, and LEDs 1430, shown in FIG. 13. In FIG. 14A, the lid 1400 isrecessed to allow mechanical fitting of the LED reflector, to preventlight escape from the well, and to enable the LEDs and reflector to bemoved closer to the target tissue or cells at the bottom of the MEAwell. The lid is furthermore shaped with a lens to refract light towardsthe center of the well, enhancing light delivery to the sample. In 14Bthe lid is shaped into a Fresnel lens, to provide similar refraction oflight, while not extending as far into the well. In 14C the lid isrecessed to a greater extent and the LEDs or LED reflector to extendmore deeply into the well, offering the potential for improved lightdelivery or light delivery concentrated towards a region of the targettissue.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the disclosed multiwellmicroelectrode arrays with integrated optical stimulation capabilitiesand associated methods for using the same. Other embodiments of thepresent disclosure will be apparent to those skilled in the art fromconsideration of the specification and practice of the presentdisclosure. It is intended that the specification and examples beconsidered as exemplary only, with a true scope of the presentdisclosure being indicated by the following claims and theirequivalents.

What is claimed is:
 1. An electro-optical stimulation and recordingsystem, comprising: a substrate; a plurality of wells coupled to thesubstrate; at least one electrode set disposed proximate a respectiveone of the plurality of wells, wherein the electrode set comprises atleast one electrode configured to collect an electric signal associatedwith at least a portion of the respective well; at least onelight-emitting element set corresponding to the respective one of theplurality of wells and configured to deliver optical stimulation to atleast a portion of the respective well.
 2. The system of claim 1,wherein the at least one electrode is further configured to deliverelectric stimulation to at least a portion of the respective well. 3.The system of claim 1, wherein the at least one electrode set furthercomprises at least one electrode configured to deliver electricstimulation to at least a portion of the respective well.
 4. The systemof claim 1, further comprising a plurality of light-emitting elementsets, wherein at least a first of the light-emitting element sets isconfigured to be illuminated independently of at least a second of thelight-emitting element sets.
 5. The system of claim 1, wherein the atleast one light-emitting element set is disposed within a wall of therespective well, disposed within the substrate, disposed within a lidconfigured to couple to the respective well, disposed within the bottomof the respective well, disposed between the substrate and therespective well, disposed beneath the respective well, or disposed abovethe respective well.
 6. The system of claim 1, wherein the plurality ofwells and the substrate from a multiwell plate, and wherein the at leastone light-emitting element set is operably coupled to an opticalstimulation module, said optical stimulation module configured todetachably couple to the multiwell plate.
 7. The system of claim 6,wherein the optical stimulation module is configured to couple to themultiwell plate such that the optical stimulation module is locatedabove the multiwell plate.
 8. The system of claim 6, wherein thesubstrate is transparent, and wherein the optical stimulation module isconfigured to couple to the multiwell plate such that it providesoptical stimulation via the base of the respective well.
 9. The systemof claim 8, further comprising a lid disposed between the at least onelight-emitting element set and the plurality of wells, wherein the lidis coated with reflective material.
 10. The system of claim 6, whereinthe at least one light-emitting element set is integrated into anelectrophysiological recording system.
 11. The system of claim 6,wherein the optical stimulation module comprises a heat sink fordissipating heat.
 12. The system of claim 1, further comprising atransparent, electrically conductive layer of indium tin oxide (ITO)between the at least one light-emitting element set and the respectivewell.
 13. The system of claim 1, further comprising a lid disposedbetween the at least one light-emitting element set and the plurality ofwells.
 14. The system of claim 13, wherein the lid comprises a pluralityof lenses configured to influence light traveling through the lid. 15.The system of claim 1, wherein at least one of the plurality of wellscomprises curved walls and/or reflective material configured toconcentrate light, shape light, and/or block bleed-through of lightbetween adjacent wells.
 16. The system of claim 1, wherein the at leastone light-emitting element set provides a wavelength and light intensitysuitable for activating opsins used in optogenetics methodologies.
 17. Amethod for large-scale in vitro testing of cell cultures, the methodcomprising: providing a control signal for causing a light-emittingelement to emit light in an illumination pattern, the light-emittingelement disposed proximate a well of a multiwell plate; detecting, viaan electrode disposed proximate the well, a signal associated with atleast a portion of the well; analyzing the detected signal; andoutputting information indicative of the analysis.
 18. The method ofclaim 17, further comprising: comparing the detected signal withbenchmark data; determining, based on the comparison, that adjustment ofthe illumination pattern is required; and modifying the illuminationpattern based on the determination.
 19. The method of claim 17, whereinthe illumination pattern includes at least one of an amplitude of alight signal, a wavelength of a light signal, and a configuration of apulse train associated with a light signal delivered to the well. 20.The method of claim 17, wherein the control signal is configured toexcite opsins of a sample within the well to the extent necessary toalter cell activity or excitability, change protein expression, and/oralter cellular growth or development.
 21. An electro-optical stimulationand recording system, comprising: an electrode set disposed proximate afirst well and comprising a first electrode configured to collect anelectric signal associated with at least a portion of the first well; afirst light-emitting element set configured to deliver opticalstimulation to at least a portion of the first well; and a processor,configured to: provide a first control signal for causing a firstlight-emitting element to emit light at a first illumination pattern;and detect, via the first electrode, a first signal associated with atleast a portion of the first well.
 22. The system of claim 21, whereinthe processor is further configured to: provide a second control signalfor causing a second light-emitting element to emit light at a secondillumination pattern, wherein the second light-emitting element isdisposed proximate a second well, and wherein the first and secondlight-emitting elements are configured to be illuminated independentlyof one another; and detect, via a second electrode, a second signalassociated with at least a portion of the second well.
 23. The system ofclaim 21, wherein the processor is further configured to: compare thedetected first signal with respective first benchmark data; determine,based on the comparison, that adjustment of the first illuminationpattern is required; and modify the first illumination pattern based onthe determination.
 24. The system of claim 21, wherein the processor isfurther configured to display, on a user interface, a representation ofthe detected first signal.
 25. The system of claim 21, wherein theprocessor is further configured to display, on a user interface, arepresentation of the first control signal.
 26. The system of claim 21,wherein the processor is further configured to display, on a userinterface, representations of the first control signal and the detectedfirst signal.
 27. The system of claim 21, wherein the processor isfurther configured to display, on a user interface, representations ofthe first control signal corresponding to a color, intensity, duration,timing, and/or location of light emitted with respect to the first well.28. The system of claim 21, wherein the processor is further configuredto update optical stimulation parameters based on metrics derived fromthe detected first signal.